1. Field of the Invention
The invention relates to aqueous L-lysine-containing animal feed supplements, which optionally contain preferably inactivated microorganisms from the fermentation process, and a process for the production of such supplements.
2. Background Information
The essential amino acid L-lysine is widely used as an animal feed supplement.
It is known that L-lysine is produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to its great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to the intrinsic performance characteristics of the microorganisms by for example mutagenesis and selection, to measures concerning fermentation technology, for example stirring and oxygen supply, to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up to the product presentation by, for example, ion exchange chromatography or crystallization.
According to the prior art, there are three different groups of L-lysine-containing product presentations which are produced from L-lysine-containing fermentation broths. The best known group comprises pulverulent or crystalline product presentations obtained from cleaned-up or pure L-lysine, which is typically present in the form of a salt such as, for example, L-lysine monohydrochloride. Another group of solid product presentations, such as are described in EP-B-0533039, for example, contains, in addition to L-lysine, the feedstock used during fermentative production and optionally the inactivated biomass of the microorganism used.
Pulverulent, crystalline or indeed granular product presentations exhibit clear disadvantages with regard to handling properties and application. The quality of the product may be influenced to a considerable extent by the weather, for example, high air humidity levels, since the caking which may occur as a consequence of such atmospheric influences impairs the shelf life and dispensing properties of the product when used. Processing of dry, pulverulent L-lysine-containing products may lead, in turn to undesirable dust development.
The third group of L-lysine-containing products comprises concentrated, aqueous, alkaline solutions and thereby circumvents the above-mentioned problems (EP-B-0534865). Given the various product presentations, many different processes are known for producing L-lysine-containing products from L-lysine-containing fermentation broths. There are essentially two different processes currently known for producing solid, pure L-lysine.
Lysine may be obtained as a monohydrochloride (Lys-HCl) by crystallization from the corresponding fermentation broths, after the inactivated biomass has been separated off by suitable methods. Cleaning-up of the filtrate prior to further concentration is performed in general by ion exchange chromatography over several stages. First of all, the fermentation broth separated from the biomass is acidified, preferably by the addition of hydrochloric acid (HCl) or sulfuric acid (H2SO4), to ease adsorption of the lysine on the ion-exchange resins. In addition to the L-lysine produced by fermentation, various other cations which are present in the fermentation broth are also bound. In general, various ion-exchange columns connected in sequence are necessary for obtaining a pure product. The adsorbed lysine is then preferably eluted by an ammoniacal solution and the ion-exchange column is regenerated. The lysine solution obtained in this way is then concentrated and lysine-HCl is obtained in crystalline form after neutralization with hydrochloric acid.
Another method enables lysine to be obtained in the form of a crystalline salt after purifying with activated carbon (SU-183581). The lysine-containing fermentation broth is inactivated by standard processes using moist heat and separated off from the biomass by filtration. After acidification of the filtrate to pH 5, 4-5% activated carbon is added with constant stirring at 50-55xc2x0 C., in order to separate off undesirable impurities from the filtrate and to prevent discoloration of the crystallizate. In a further filtration stage thereafter, the activated carbon is separated off and the dissolved sulfate is then precipitated as calcium sulfate by the addition of calcium hydroxide. This is filtered off, the ammonia content being removed in a rotary evaporator under vacuum and the solution being concentrated until crystallization occurs on cooling.
The disadvantage of these two preparation methods lies in the numerous individual stages and the complex cleaning processes using ion-exchange chromatography. The elimination of troublesome salts or the use of different elution media creates additional waste streams, which have either to be cleaned by complex methods or expensively disposed of.
EP-B-0533039 counters these disadvantages in that all the fermentation feedstock, optionally including the biomass, is concentrated and spray-dried at elevated temperature. U.S. Pat. No. 5,990,350 describes a process for producing biomass-free L-lysine-containing granules. In this process, the biomass is first isolated from the fermentation broth by ultrafiltration and discarded. The filtrate recovered in this way is then concentrated by extensive evaporation of the water content. The concentrate obtained in this way is finally dried and worked up in a complex manner by fluidised bed spray granulation to produce granules. A process is known from EP-B-0534865 for producing aqueous, basic, L-lysine-containing solutions from fermentation broths. In the process described therein, the biomass is separated from the fermentation broth and discarded. A pH value of between 9 and 11 is established using a base such as sodium, potassium or ammonium hydroxide, for example. The mineral constituents (inorganic salts) are separated from the broth by crystallization after concentration and cooling and either used as fertiliser or discarded (land-filled).
The object of the invention consists in providing novel, aqueous preparations of L-lysine and its salts suitable as a feed supplement. In addition, it is an object of the invention to provide a production process that is more economical and efficient than processes currently known.
The essential amino acid lysine is widely used as an animal feed supplement.
Microorganisms of the genus Corynebacterium are distinguished by the capacity to secrete high concentrations of L-lysine into the corresponding fermentation medium over a relatively short period. The production processes are generally performed as fed batch processes. L-lysine produced by fermentation is currently predominantly worked up to produce a crystalline product, powder or granules.
The present invention provides a fermentation broth-based animal feed supplement, characterized in that it contains
a) L-lysine and/or salts thereof,
b) the biomass formed during fermentation in an amount of from 0 to 100%, preferably from 50 to 100% and very particularly from 90 to 100%,
c) at least the majority of the other dissolved and suspended constituents of the fermentation broth are present in liquid form, optionally as a suspension and
e) has a pH of less than 7.
The animal feed supplement has a lysine content (as lysine base) of 10 wt. % to 35 wt. %, preferably 15 wt. % to 35 wt. %, particularly preferably 18 wt. % to 35 wt. % and very particularly preferably 21 wt. % to 34 wt. %, relative to the total amount of supplement. The pH is less than 7, preferably 2 to 6.5, particularly preferably 2.5 to 6 and very particularly preferably 2.5 to 5.
The total solids content of the supplement amounts to from 10 wt. % to 55 wt. %, preferably 20 wt. % to 55 wt. %, particularly preferably 35 wt. % to 55 wt. % and very particularly preferably 45 wt. % to 54 wt. %. The solids content contains, in addition to lysine
preferably the biomass of the producing microorganism,
the inorganic and other organic constituents of the fermentation broth and
the by-products produced during fermentation,
insofar as they have not been separated off by suitable processes such as, for example, separation or filtration. If the biomass is left completely or partially in the supplement, the latter contains the microorganism protein in a concentration of up to a maximum of 4 wt. %. The supplement contains as inorganic constituents inter alia calcium, magnesium, phosphorus in the form of phosphate and sulfur in the form of sulfate and as organic constituents inter alia vitamins, such as biotin and thiamin, for example, or sugar, such as isomaltose, for example.
The organic by-products formed in small amounts during fermentation include L-amino acids selected from the group comprising L-alanine, L-asparagine, L-glutamine, L-methionine, L-threonine and L-valine. They additionally include organic acids which carry one to three carboxyl groups, such as for example lactic acid, acetic acid and malic acid. Finally, they also include sugars, such as trehalose, for example. These compounds may be desirable if they improve the valency of the supplement.
The invention further provides a process for producing aqueous solutions/suspensions containing lysine and/or the salts thereof, which process is characterized in that
a) an L-lysine-containing broth is produced by fermentation,
b) the desired lysine concentration is optionally established by the addition of an L-lysine-containing substance,
c) the pH value of the solution/suspension is adjusted to  less than 7, and
d) the broth obtained in this way is optionally concentrated under vacuum,
wherein the order of the steps b), c) and d) may vary.
Finally, the invention further provides a process for producing aqueous solutions/suspensions containing lysine and/or salts thereof, which process is characterized in that
a) an L-lysine-containing broth is produced by fermentation,
b) the pH value of the broth obtained in this way is adjusted to less than 7,
c) the biomass is optionally separated off completely or partially from the solution/suspension,
d) the desired lysine concentration is optionally established by the addition of an L-lysine-containing substance,
e) and the broth obtained in this way is optionally concentrated under vacuum,
wherein the order of the steps b), c), d) and e) may vary.
Mutant coryneform bacteria producing L-lysine are described in detail in the prior art, such as for example in U.S. Pat. No. 4,657,860.
For the purpose of L-lysine production, these strains may be cultured continuously or discontinuously using the batch process or the fed batch process or the repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel (Dioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must adequately satisfy the requirements of the particular strains. Carbon sources which may be used are sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose for example, oils and fats, such as soya oil, sunflower oil, peanut oil and coconut oil for example, fatty acids, such as palmitic acid, stearic acid and linoleic acid for example, alcohols, such as glycerol and ethanol for example, and organic acids, such as acetic acid for example. These substances may be used individually or as a mixture. Nitrogen sources which may be used are organic, nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used are potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium has additionally to contain salts of metals, such as magnesium sulfate or iron sulfate for example, which are necessary for growth. Finally, essential growth-promoting substances, such as amino acids and vitamins, may be used in addition to the above-mentioned substances. Suitable precursors may furthermore be added to the culture medium. The stated feedstock substances may be added to the culture as a single batch or be fed appropriately during cultivation.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Foaming may be controlled by using antifoaming agents such as fatty acid polyglycol esters for example. Plasmid stability may be maintained by the addition to the medium of suitable selectively acting substances, for example antibiotics. The introduction of oxygen or oxygen-containing gas mixtures such as air into the culture and thorough mixing using suitable stirring systems or the gas stream may be used to maintain aerobic conditions. The temperature of the culture is typically 25xc2x0 C. to 37xc2x0 C. The culture is continued until the maximum quantity of L-lysine has formed. This aim is normally achieved within 10 to 160 hours.
Examples of suitable fermentation media may be found, for example, in patent specifications EP-B-0 532 867, U.S. Pat. No. 5,840,551 and U.S. Pat. No. 5,990,350.
Analysis of L-lysine may be performed by anion exchange chromatography with subsequent ninhydrin derivatisation, as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) or it may be performed by reversed phase HPLC, as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
The fermentation broths used for the process according to the invention preferably have an L-lysine content greater than 60 g/L (as lysine base) for a content of non-metabolised sugar of less than 5.0 g/L. Out of a total solids content of  greater than 10 wt. %, the biomass preferably accounts for 1 to 4 wt. %. The content of by-products and vitamins from fermentation (amino acids, organic acids) is preferably less than 2 wt. %.
In the process according to the invention, the biomass present in the fermentation broth is generally inactivated or destroyed at the start, for example by heat treatment. Inactivation may optionally be dispensed with, however. A pH value of less than 7, preferably 2 to 6.5, particularly preferably 2.5 to 6 and very particularly preferably 2.5 to 5 is then established in the fermentation broth by means of an inorganic acid, such as sulfuric acid, hydrochloric acid or phosphoric acid for example, or an organic acid, such as citric acid, acetic acid or formic acid for example, or a mixture of different acids. The biomass is optionally separated off completely or partially by generally known separation or filtration methods before or after acidification. It is not generally necessary to separate off the mineral constituents. The acidic L-lysine-containing solution or suspension is then concentrated using known methods (such as for example a rotary evaporator, film evaporator or falling-film evaporator), preferably under vacuum, until a liquid product is obtained with a lysine content (as lysine base) of 10 wt. % to 35 wt. %, preferably 15 wt. to 35 wt. %, particularly preferably 20 wt. % to 35 wt. % and very particularly preferably 21 wt. % to 34 wt. %, for a total solids content of 10 wt. % to 55 wt. %, preferably 20 wt. % to 55 wt. %, particularly preferably 35 wt. % to 55 wt. % and very particularly preferably 45 wt. % to 54 wt. %. It is generally the case that the broth is so concentrated that preferably no mineral constituents (inorganic salts) precipitate out of the fermentation broth and the lysine is present in solution. A desired concentration of L-lysine may optionally be established in the product by the addition of an L-lysine-containing substance at any process stage. The solution/suspension obtained in this way has an acid pH value, is easy to transport, may be readily dispensed, is microbially stable and has a longer shelf life than an alkaline solution.
Acidification may also be performed after or during concentration. The anions acting as sulfate, chloride, phosphate, citrate etc. may be added to the medium in the form of commercially available salts even prior to fermentation.
The term xe2x80x9csuspensionxe2x80x9d is used to indicate that the preferably inactivated microorganisms are present in the product according to the invention in undissolved form.